Childhood herpes simplex encephalitis (HSE) is a life-threatening complication of primary infection by herpes simplex virus 1 (HSV-1), a common virus that is typically innocuous. HSE is the most common sporadic viral encephalitis in Western countries and acyclovir-treated survivors often suffer from severe neurological sequelae. The pathogenesis of HSE remained unclear until we showed that the disease results, in some children, from single-gene mutations impairing TLR3- and IFN-a/-mediated immunity to HSV-1 in the central nervous system (CNS). Following a candidate gene approach, we reported 13 patients, each carrying rare mutations in one TLR3 pathway gene (TLR3, UNC93B1, TRIF, TRAF3, TBK1). We also demonstrated that patient-specific induced pluripotent stem cell (iPSC)-derived TLR3-deficient neurons and oligodendrocytes are susceptible to HSV-1 infection, suggesting that impaired TLR3- and IFN-a/-mediated CNS-intrinsic anti-HSV- 1 immunity underlies the pathogenesis of HSE in these patients. In parallel, thanks to NIH R01A1088364, we initiated a hypothesis-generating search for novel HSE-causing candidate genes in consanguineous HSE kindreds by genome-wide (GW) linkage (GWL). We discovered DBR1 as a novel HSE-causing gene in two relatives. However, no genetic etiology has yet been identified for 235 of the 250 HSE patients under study. We now hypothesize that HSE in some of these children is a consequence of a collection of CNS-intrinsic inborn errors of immunity to HSV-1, possibly but not necessarily related to the TLR3-IFN-a/ circuit. The goal of this renewal application is to extend the GW approach by taking advantage of whole-exome sequencing (WES) to study not only consanguineous (28 patients), but also non-consanguineous (207) patients (trio design). We will analyze WES data in two ways: 1) hypothesis-based, searching for mutations in TLR3-IFN-a/ pathway genes; 2) hypothesis-generating, searching for mutations in other genes. We will benefit from GWL and human gene connectome analysis. We will consider models of genetic homogeneity and heterogeneity. Whole-genome sequencing (WGS) will be performed in patients for whom WES fails to reveal candidate mutations. We will use patients' fibroblasts to investigate the impact of the new candidate genetic etiologies on anti-HSV-1 immunity. This application is innovative but supported by strong evidence. We have established a unique international cohort of 250 HSE children. A hypothesis-based analysis of the WES data for 170 HSE patients has already revealed non-synonymous rare mutations of 18 key genes of the TLR3-IFN pathway in up to 52 patients. Unbiased GW analysis revealed non-synonymous rare heterozygous mutations in the IFN-inducible gene IFIT2 in 4 patients, and in a small non-coding RNA gene SNORA31 in 6 other patients. Our research will decipher the pathogenesis of a devastating pediatric illness, paving the way for new therapeutic approaches. The genetic analysis of HSE will also provide proof-of-principle that sporadic, life-threatening infectious diseases in otherwise healthy children may result from single-gene inborn errors of immunity.